We describe the construction and characterization of a series of novel cyclin-dependent kinase inhibitors with increased antiproliferative activity for use in the genetic treatment of hyperproliferative cell disorders, such as angioplasty-induced restenosis. These inhibitors were generated through the fusion of truncated versions of the p27 gene to the full-length p16 gene. Biochemically, the p27-p16 chimeric molecules were of comparable potency to the parental p27 in inhibiting the activities of several cyclin-dependent kinases in vitro. Replication-deficient adenoviruses encoding the parental p16, p27 genes, or their derivatives were created to assess the potency of the novel cyclin-dependent kinase inhibitor chimeric molecules to inhibit vascular smooth muscle cell proliferation, which is the seminal event in the restenosis process. One of the p27-p16 chimeric molecules, W9, was observed to be the most potent inhibitor of human primary smooth muscle and endothelial cell proliferation when compared to the p16, p27, p27 derivatives or several alternative p27-p16 chimeric molecules. Overexpression of the W9 chimeric molecule in human coronary artery smooth muscle cells induced human coronary artery smooth muscle cell growth arrest in G1 but did not induce cell apoptosis. Recombinant adenoviral vectors that express this W9 chimeric cyclin-dependent kinase inhibitor molecule constitute a novel potent antiproliferative agent for the treatment of restenosis.